Chondrocyte-like cell, and method for producing same

ABSTRACT

Disclosed is a cell which enables the reproduction of a cartilage tissue and has a proliferative ability. Also disclosed is a technique for providing a cell supply source which can be used in a definitive treatment of osteochondrosis deformans. A chondrocyte-like cell which has the same properties as those of a chondrocyte and can proliferate can be produced by selecting a combination of an Myc family gene and/or a Klf family gene and a SOX9 gene and introducing the combination into a somatic cell. The chondrocyte-like cell can be used for a medical purpose of cartilage regeneration.

TECHNICAL FIELD

The present invention relates to chondrocyte-like cells that are inducedfrom somatic cells and have proliferative capabilities and theproperties of the chondrocytes, and to processes for producing thechondrocyte-like cells. The invention also concerns cell preparationsfor cartilage tissue regeneration, implants, implant producingprocesses, cartilage disease therapeutic methods, and drug efficacydetermining methods for determining the efficacy of a tested substancefor cartilage disease, all using the chondrocyte-like cells. Theinvention also relates to chondrocyte-like cell preparation compositionsused to induce somatic cells to the chondrocyte-like cells.

BACKGROUND ART

Articular cartilage has a role as a joint lubricant for absorbing impactat the diarthrodial joints during articular movement. The mechanicalfunctions of the cartilage are imparted by the cartilage extracellularmatrix constructed from type II and type XI collagens, and collagenousfibrils such as proteoglycan. It is known that the cartilageextracellular matrix is produced by the chondrocytes intrinsic to thecartilage.

Osteoarthritis is a typical cartilage tissue disease, caused by theaggravation of wear, damage, and degeneration of the articular cartilagein response to mechanical stresses (such as repetitive loading,excessive exercise, and trauma) and aging. The symptoms ofosteoarthritis include joint pain during joint movement (movement pain)and a restricted range of motion (restricted motion), which lower thequality of daily life. In Japan, osteoarthritis affects about 20% of thepopulation over the age 50, and is expected to affect more people as themedical development and improved lifestyle are expected to raise theaverage life expectancy. Osteoarthritis thus poses a big challenge inthe aging society.

Conventional osteoarthritis therapies employ resting to preventaggravation of symptoms or controlling pain by, for example, theadministration of antiphlogistic analgetics or supplements, or theintraarticular administration of joint lubricants. These methods,however, are only supportive, and do not represent a definitive therapy,because the chondrocytes have only weak repairing capabilities (seeNon-Patent Literature 1), and cannot regenerate cartilage tissue. Aprocedure using a metallic artificial replacement joint has beenpracticed for osteoarthritis cases with progressive cartilagedegeneration. However, artificial joints have a number of drawbacks,including a heavy burden put on patients during the procedure,deterioration due to wear, a tendency to dislocate, and possiblerevision surgery necessitated by a loosened artificial joint.

Recently, a technique that enables a definitive treatment ofosteochondrosis deformans through cartilage tissue regeneration hascaught attention for the treatment of osteochondrosis deformans whichdoes not respond well to conventional therapies. For such a technique tobe realized, development of an easy-to-obtain cell supply source thatcan produce large numbers of cells while retaining the capability todifferentiate and form cartilage tissue is urgently needed (seeNon-Patent Literatures 2 and 3). Chondrocytes are considered as a goodcandidate for such a cell supply source used for cartilage tissueregeneration. However, because chondrocytes are limited in number andcause dedifferentiation through monolayer expansion (see Non-PatentLiterature 4), recent studies focus more on the development of atechnique that induces formation of cartilage tissue with the use ofbone marrow-derived mesenchymal stem (MS) cells or embryonic stem (ES)cells (see Non-Patent Literatures 5 to 7). However, MS cells have onlylimited proliferative capabilities, and recent studies suggest that thecartilage produced from MS cells is unstable, and lacks sufficientcartilage properties (see Non-Patent Literatures 8 and 9). With regardto ES cell-derived differentiated cells, there are concerns that thecells, as an inhomogeneous population, may fail to provide sufficientcartilage tissue functions (see Non-Patent Literatures 10 and 11), ormay cause formation of teratoid tumors (see Non-Patent Literature 12).

There are also reports of a technique that has brought innovation in thefield of regenerative medicine, specifically a technique that reprogramsand induces somatic cells to induced pluripotent stem (iPS) cells byintroducing Oct3/4, Klf4, c-Myc, and Sox2 coding genes into the somaticcells (see Patent Literature 1, Non-Patent Literatures 13 to 24).However, because of the pluripotency of iPS cells, use of iPS cells forcartilage tissue regeneration requires the establishment of a techniquethat enables the cells to differentiate into a homogeneous chondrocytepopulation, and there are still technical problems that need to besolved for practical applications in cartilage tissue regeneration.

Over these backgrounds, there is a growing need for the development ofcells that can be directly induced to only chondrocytes, and that havecartilage tissue regenerative capabilities and a proliferative ability,and for a cell supply source that can also be used for a definitivetreatment of osteochondrosis deformans.

CITATION LIST Patent Literature

-   PTL 1: International Publication 2007/069666

Non-Patent Literature

-   NPL 1: W. Hunter, Philos Trans Lond 42, 514 (1743).-   NPL 2: C. Chung and J. A. Burdick, Adv Drug Deliv Rev 60 (2), 243    (2008).-   NPL 3: J. Gao, J. Q. Yao, and A. I. Caplan, Proc. Inst. Mech. Eng.    [H]. 221 (5), 441 (2007).-   NPL 4: U. R. Goessler, P. Bugert, K. Bieback et al., Int. J. Mol.    Med. 14 (6), 1015 (2004).-   NPL 5: J. Kramer, C. Hegert, K. Guan et al., Mech. Dev. 92 (2), 193    (2000).-   NPL 6: N. S. Hwang, M. S. Kim, S. Sampattavanich et al., Stem Cells    24 (2), 284 (2006).-   NPL 7: N. S. Hwang, S. Varghese, and J. Elisseeff, PLoS ONE 3 (6),    e2498 (2008).-   NPL 8: V. Vacanti, E. Kong, G. Suzuki et al., J. Cell. Physiol.    205(2), 194 (2005).-   NPL 9: A. Nagai, W. K. Kim, H. J. Lee et al., PLoS ONE 2 (12), e1272    (2007).-   NPL 10: M. Arndt and J. Itskovitz-Eldor, Journal of anatomy 200    (Pt3), 225 (2002).-   NPL 11: E. J. Koay, G. M. Hoben, and K. A. Athanasiou, Stem Cells 25    (9), 2183 (2007).-   NPL 12: S. Wakitani, K. Takaoka, T. Hattori et al., Rheumatology    (Oxford). 42 (1), 162 (2003).-   NPL 13: T. Aoi, K. Yae, M. Nakagawa et al., Science 321 (5889), 699    (2008).-   NPL 14: M. Nakagawa, M. Koyanagi, K. Tanabe et al., Nat. Biotechnol.    26 (1), 101 (2008).-   NPL 15: K. Takahashi, K. Okita, M. Nakagawa et al., Nature protocols    2 (12), 3081 (2007).-   NPL 16: K. Takahashi, K. Tanabe, M. Ohnuki et al., Cell 131 (5), 861    (2007).-   NPL 17: K. Takahashi and S. Yamanaka, Cell 126 (4), 663 (2006).-   NPL 18: K. Okita, T. Ichisaka, and S. Yamanaka, Nature 448 (7151),    313 (2007).-   NPL 19: M. Wernig, A. Meissner, R. Foreman et al., Nature 448    (7151), 318 (2007).-   NPL 20: N. Maherali, R. Sridharan, W. Xie et al., Cell stem cell    1(1), 55 (2007).-   NPL 21: A. Meissner, M. Wernig, and R. Jaenisch, Nat. Biotechnol. 25    (10), 1177 (2007).-   NPL 22: M. Wernig, A. Meissner, J. P. Cassady et al., Cell stem cell    2 (1), 10 (2008).-   NPL 23: J. Yu, M. A. Vodyanik, K. Smuga-Otto et al., Science 318    (5858), 1917 (2007).-   NPL 24: I. H. Park, R. Zhao, J. A. West et al., Nature 451 (7175),    141 (2008).

SUMMARY OF INVENTION Technical Problem

It is an object of the present invention to solve the foregoingtechnical problems. Specifically, an object of the present invention isto establish a technique for developing cells that have cartilage tissueregenerative capabilities and a proliferative ability, and for providinga cell supply source that can also be used for a definitive treatment ofosteochondrosis deformans.

Solution to Problem

The present inventors conducted intensive studies to solve the foregoingproblems, and found that chondrocyte-like cells that have aproliferative ability and the properties of the chondrocytes could beproduced by introducing into somatic cells a combination of Myc familygene and/or Klf family gene and SOX9 gene selected from large numbers ofdifferentiating cell reprogramming factors and cartilage-associatedgenes. It was confirmed that the chondrocyte-like cells actuallyobtained were able to proliferate in a monolayer culture, and expressedcartilage specific markers. It was also confirmed that thechondrocyte-like cells were able to form a cartilage tissue whencultured using a collagen gel as a scaffold, or when administered to anorganism without using a scaffold. The present invention was completedbased on these findings upon further studies.

Specifically, the present invention provides the following aspects ofinvention.

Item 1.

A chondrocyte-like cell producing process including the step ofintroducing into a somatic cell a SOX9 gene and at least one geneselected from the group consisting of Myc family gene and Klf familygene.

Item 2.

A producing process according to Item 1, wherein the Myc family gene isa c-Myc gene.

Item 3.

A producing process according to Item 1 or 2, wherein the Klf familygene is a Klf4 gene.

Item 4.

A producing process according to any one of Items 1 to 3, wherein thesomatic cell originates in humans.

Item 5.

A producing process according to Item 1 or 2, wherein the somatic cellis a dermal fibroblast or an adipose tissue-derived stromal cell.

Item 6.

A chondrocyte-like cell obtained by introducing into a somatic cell aSOX9 gene and at least one gene selected from the group consisting ofMyc family gene and Klf family gene.

Item 7.

A chondrocyte-like cell according to Item 6, wherein the Myc family geneis a c-Myc gene.

Item 8.

A chondrocyte-like cell according to Item 6 or 7, wherein the Klf familygene is a Klf4 gene.

Item 9.

A chondrocyte-like cell according to any one of Items 6 to 8, whereinthe somatic cell originates in humans.

Item 10.

A chondrocyte-like cell according to any one of Items 6 to 9, whereinthe somatic cell is a dermal fibroblast or an adipose tissue-derivedstromal cell.

Item 11.

A cell preparation for cartilage tissue regeneration, including thechondrocyte-like cell of any one of Items 6 to 9.

Item 12.

A cell preparation according to Item 11, wherein the cell preparationincludes a scaffolding material.

Item 13.

A cell preparation according to Item 12, wherein the scaffoldingmaterial is a collagen.

Item 14.

An implant including a cartilage tissue constructed by using thechondrocyte-like cell of any one of Items 6 to 8.

Item 15.

A process for producing a cartilage tissue implant,

the process including the steps of:

-   -   administering the chondrocyte-like cell of any one of Items 6 to        8 into a body of a mammal; and    -   removing a cartilage tissue formed from the chondrocyte-like        cell in the body of the mammal.

Item 16.

A cartilage disease therapeutic method,

the method including the steps of:

-   -   administering the chondrocyte-like cell of any one of Items 6 to        8 into a cartilage disease patient at a non-cartilage tissue        site; and    -   removing a cartilage tissue formed from the chondrocyte-like        cell, and transplanting the removed cartilage tissue to the        cartilage disease site of the patient.

Item 17.

A use of the chondrocyte-like cell of any one of Items 6 to 9 for theproduction of a cell preparation for cartilage tissue regeneration.

Item 18.

A use according to Item 17, wherein the cell preparation for cartilagetissue regeneration is a therapeutic agent for cartilage disease.

Item 19.

A use of a composition containing the chondrocyte-like cell of any oneof Items 6 to 9 and a scaffolding material for the production of a cellpreparation for cartilage tissue regeneration.

Item 20.

A use according to Item 19, wherein the scaffolding material is acollagen.

Item 21.

A non-human mammal forming a cartilage tissue, wherein the non-humanmammal is produced by administering the chondrocyte-like cell of any oneof Items 6 to 9 into a non-human mammal, and by forming a cartilagetissue from the chondrocyte-like cell in a body of the mammal.

Item 22.

A method for determining the efficacy of a tested substance for acartilage tissue,

-   -   the method including the step of administering the tested        substance to the non-human mammal of Item 21 and determining the        efficacy of the tested substance for the cartilage tissue.

Item 23.

A chondrocyte-like cell preparation composition, including a SOX9 geneand at least one gene selected from the group consisting of Myc familygene and Klf family gene.

Item 24.

A chondrocyte-like cell preparation composition according to Item 23,wherein a SOX9 gene and at least one gene selected from the groupconsisting of Myc family gene and Klf family gene are contained in aform introducible to a somatic cell.

Advantageous Effects of Invention

The present invention provides chondrocyte-like cells that have aproliferative ability and the properties of the chondrocytes, and thuscan provide a medical means effective for the treatment of cartilagedisease that involve cartilage damage such as in osteochondrosisdeformans. Further, the present invention enables production ofchondrocyte-like cells from the somatic cells of patients suffering fromosteoarthritis and a wide range of other cartilage diseases including agrowth cartilage disease such as chondrodystrophy, and can thuscontribute to elucidating the pathology of the disease by performingvarious analyses. The chondrocyte-like cells produced from humans areparticularly suitable as material for the discovery and development ofdrugs.

Further, because the present invention can be used to obtainchondrocyte-like cells from skin tissue-derived somatic cells such asskin fibroblasts and subcutaneous adipose tissue-derived stromal cells,the invention is highly useful in the clinic from the standpoint ofreducing burdens on patients and cell donors.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram representing a Col11a2-β geo transgenic mouse, andthe results of evaluations on the properties of the primarychondrocytes, MEFs, and MDFs isolated from the mouse; a, a configurationof a gene introduced into the transgenic mouse; b (left), an image of aCol11a2-β geo transgenic mouse stained with X-gal; b (right), the tissueanalysis result for the cartilage of the X-gal-stained Col11a2-β geotransgenic mouse; c (left), the primary chondrocytes prepared from the βgeo transgenic mouse, observed under a phase-contrast microscope; c(right), the result of the X-gal staining of the primary chondrocytesprepared from the β geo transgenic mouse; d, the results of incubatingthe primary chondrocytes, MEFs, and MDFs prepared from the β geotransgenic mouse, and the primary chondrocytes prepared from wild-typeF1 hybrid mouse in the presence of 0 to 900 μg/ml G418, in which “Tg”means cells of β geo transgenic mouse origin, and “WT” means cells ofwild-type littermate mouse origin; the same denotation is also used inthe other figures in the same meaning.

FIG. 2 is a diagram representing the analysis results for the cellsproduced by introducing each factor into MEFs; a, the number of stainedcolonies counted after the alucian blue staining and crystal violetstaining of the colonies obtained by introducing each factor into MEFs,in which “4R” represents four reprogramming factors (Oct3/4, Sox2,c-Myc, and Klf-4), also used in the other figures in the same meaning;b, the results of the alucian blue staining and crystal violet stainingof the cells obtained by transfecting MEFs with the four reprogrammingfactors (Oct3/4, Sox2, c-Myc, and Klf-4) in combination with human SOX9;c, the results of observing the shape of the cells contained in thecolonies obtained by transfecting MEFs with the four reprogrammingfactors (Oct3/4, Sox2, c-Myc, and Klf-4) in combination with human SOX9;d, the result of observing MEF shape; e, the number of colonies countedafter the alucian blue staining and crystal violet staining of thecolonies obtained by introducing three reprogramming factors and Sox9,in which “4R-c-Myc” means the four reprogramming factors (Oct3/4, Sox2,c-Myc, and Klf-4) minus c-Myc, “4R-Klf4” means the four reprogrammingfactors (Oct3/4, Sox2, c-Myc, and Klf-4) minus Klf-4, “4R-Oct3/4” meansthe four reprogramming factors (Oct3/4, Sox2, c-Myc, and Klf-4) minusOct3/4, and “4R-Sox2” means the four reprogramming factors (Oct3/4,Sox2, c-Myc, and Klf-4) minus Sox2; the same denotation is also used inthe other figures in the same meaning; f, the results of observing theshape of the cells contained in the colonies obtained by transfectingMEFs with c-Myc, Klf4, and Sox9.

FIG. 3 is a diagram representing the analysis results for the cellsproduced by introducing each factor into MDFs; A to C, the number ofstained colonies counted after the alucian blue staining and crystalviolet staining of the colonies obtained by introducing differentfactors to MDFs in various combinations, and the number of coloniesformed by polygonal cells; D, the results of observing the morphology ofthe cells contained in the colonies obtained by introducing each factorto MDFs; E, the results of observing the shape of each cell afterculturing the cells contained in the colonies obtained by introducingeach factor to MDFs.

FIG. 4 is a diagram representing the results of evaluating theproperties of the cells (cloned cells) obtained by introducing eachfactor to MDFs; A, the results of the alucian blue staining of MDFs andthe cells obtained by introducing each factor to MDFs; B, the analysisresults for the expression of the introduced genes (introduced factors)in MDFs, primary chondrocytes, and the cells obtained by introducingeach factor to MDFs, in which “Pr chond.” means primary chondrocytes,also used in the other figures in the same meaning; C, the results ofanalyses for the expression of the introduced genes (introduced factors)in MDFs, primary chondrocytes, and the cells obtained by introducingeach factor to MDFs.

FIG. 5 is a diagram representing the results of evaluating theproperties of the cells (cloned cells) obtained by introducing eachfactor to MDFs; a, the analysis results for the expression ofchondrocyte marker genes in MDFs, primary chondrocytes, and the cellsobtained by introducing each factor to MDFs; b, the analysis results forthe expression of chondrocyte marker genes in MDFs, primarychondrocytes, and the cells obtained by introducing each factor to MDFs;c, the results of the karyotyping of the cells (MK-4, MKO-2) obtained byintroducing each factor to MDFs.

FIG. 6 is a diagram representing the results of evaluating theproperties of the cells (cloned cells) obtained by introducing eachfactor to MDFs; a, a diagram comparing gene expression patterns forprimary chondrocytes and MDFs; b, a diagram comparing gene expressionpatterns for MK-3 and primary chondrocytes; c, a diagram comparing geneexpression patterns for MK-3 and MDFs; d, the result of the clusteranalyses of MDFs, primary chondrocytes, and the cells obtained byintroducing each factor to MDFs; e, the results of the bisulfite genomicsequencing analyses of MK-3, MK-4, and MDFs concerning the dinucleotidemethylation status, in which the solid dots represent methylated CpGdinucleotide in each gene, and open dots represent unmethylated CpGdinucleotide in each gene.

FIG. 7 represents the results of evaluating the properties of the cells(cloned cells) obtained by introducing each factor to MDFs; a, theanalysis results for the proliferative properties of MDFs and of thecells obtained by introducing each factor to MDFs; b, the results ofalucian blue staining after culturing MDFs and the cells obtained byintroducing each factor to MDFs; c, the analysis results for thegel-cell complex formed after culturing MK-3 and MDFs with collagengels.

FIG. 8 is a diagram representing nude mice after the subcutaneousinjection of cells (MK-5) obtained by introducing c-Myc, Klf-4, Sox9,and GFP to MDFs; A, the result of the observation of the whole nudemouse (right, the result of fluorescence observation); B, the result ofthe observation of a nude mouse after removing the skin on the back(right, the result of fluorescence observation); C, the result of thesafranine O staining of a continuous tissue slice obtained from thesubcutaneous site of a cell suspension-injected mouse; D, magnificationsof the rectangles shown in C.

FIG. 9 represents the results of the observation of the tissue at theinjection site of a nude mouse after the subcutaneous injection of thecells (chondrocyte-like cells) obtained by introducing c-Myc, Klf-4, andSox9 to MDFs; A, the results of the safranine O, fast green, and ironhaematoxylin staining of the tissue at the injection site after 16 weeksfrom the injection of the cells (MK-7) obtained by introducing c-Myc,Klf-4, and Sox9 to MDFs; B, the results of the safranine O, fast green,and iron haematoxylin staining of the tissue at the injection site after8 weeks from the injection of the cells (MK-10) obtained by introducingc-Myc, Klf-4, and Sox9 to MDFs.

FIG. 10 represents the result of southern hybridization performed forthe genomic DNA of each chondrocyte-like cell produced in Examples 1 and2, using Klf4 cDNA probes.

FIG. 11 represents the number of stained colonies counted after thealucian blue staining and crystal violet staining of the coloniesobtained by introducing c-Myc, Klf-4, and Sox9 to adipose tissue-derivedstromal cells, and the number of colonies formed by polygonal cells.

FIG. 12 is a diagram representing the analysis results for the cellsobtained by introducing OCT3/4, c-MYC, KLF-4, and SOX9 to NHDFs; a, theresults of alucian blue staining for the culture dish (MKO) of OCT3/4-,c-MYC-, KLF-4-, and SOX9-introduced NHDFs, and for the culture dish(EGFP) of EGFP-introduced NHDFs; b, the results of observing the shapeof the cells contained in the colonies obtained by introducing OCT3/4,c-MYC, KLF-4, and SOX9 to NHDFs; c, the results of observing the shapeof the cells in the culture dish of EGFP-introduced NHDFs; d, a diagramrepresenting the morphology of human primary chondrocytes, copied fromthe website of Cell Applications, INC.(http://www.cellapplications.com/product_desc.php?id=33&category_id=51&subcategory_id=68).

DESCRIPTION OF EMBODIMENTS 1. Chondrocyte-Like Cell Producing Process,and Use of Chondrocyte-Like Cells

As used herein, “chondrocyte-like cells” means cells that have aproliferative ability and the properties of the chondrocytes, with thecapabilities to form or regenerate cartilage tissue (in other words,cartilage stem cells). Herein, “having the properties of thechondrocytes” means showing positive with the specific staining forchondrocytes and expressing chondrocyte marker genes.

A chondrocyte-like cell producing process of the present invention is aprocess that includes the step of introducing into somatic cells atleast one gene selected from the group consisting of Myc family gene andKlf family gene, together with SOX9 gene. The producing process of thepresent invention is described below.

In the present invention, the type of somatic cells induced tochondrocyte-like cells is not particularly limited, and thoseoriginating in any tissue or site can be used. Examples of somatic cellsusable in the present invention include tissue-derived-cells such asthose from the skin, subcutaneous adipose, muscle, placenta, bone, andcartilage. More specific examples include dermal fibroblasts,subcutaneous adipose tissue-derived stromal cells (subcutaneous adiposecells), embryonic fibroblasts, adipose cells, muscle cells, osteoblasts,and chondrocytes. Of these, skin-derived cells and subcutaneous adiposecells are preferred, and skin fibroblasts and subcutaneous adiposetissue-derived stromal cells are particularly preferred, because thesecells are only mildly invasive to organism, and can more efficientlyproduce chondrocyte-like cells. Material can be selected from thesevarious types of cells, and the fact that readily available cells suchas skin-derived cells and subcutaneous adipose tissue-derived cells canbe used is particularly advantageous in clinical settings from thestandpoint of reducing the burden on patients and providing a stablecell supply. Further, the somatic cells may be commercially availableproducts, or somatic cells differentiated from cells such as ES cells ormesenchymal stem cells.

Further, the somatic cells may be appropriately selected from cells thatoriginate in mammals such as humans, mice, rats, hamsters, rabbits,cats, dogs, sheep, pigs, cows, goats, and monkeys, depending on theintended use of the chondrocyte-like cells, and cells originating inhumans are preferred for therapeutic purposes in humans. Thehuman-derived somatic cells may originate in any of fetuses, infants,children, and adults. When the chondrocyte-like cells are used fortherapeutic purposes in humans, it is desirable to use somatic cellscollected from a patient.

In the present invention, the somatic cells are induced tochondrocyte-like cells by introducing into the somatic cells at leastone gene selected as a reprogramming factor from the group consisting ofMyc family gene and Klf family gene, in combination with thecartilage-inducible transcription factor SOX9 gene.

Examples of Myc family gene include c-Myc, N-Myc, and L-Myc. The Mycfamily genes may be used either alone or in combinations of two or more.Among these Myc family genes, the present invention preferably usesc-Myc gene and L-Myc gene, more preferably c-Myc gene. c-Myc gene isknown as a transcriptional regulator involved in cell differentiationand proliferation (S. Adhikary, M. Elilers, Nat. Ray. Mol. Cell. Biol.,6, pp 635-645, 2005), and has known base sequences (NCBI accessionNumber NM_(—)010849 (human), NM_(—)002467 (Mouse)). N-Myc gene and L-Mycgene also have known base sequences (NCBI accession Number NM_(—)005378(human), NM_(—)008709 (Mouse)), and (NCBI accession Number NM_(—)005376(human), NM_(—)008506 (Mouse)), respectively. Note that NCBI in thisspecification is the abbreviation for National Center for BiotechnologyInformation.

Examples of Klf family gene include Klf1, Klf2, Klf4, and Klf5. The Klffamily genes may be used either alone or in combinations of two or more.Among these Klf family genes, the present invention preferably uses Klf2gene, Klf4 gene, and Klf5 gene, more preferably Klf2 gene and Klf4 gene,particularly preferably Klf4 gene. Klf4 gene is known as a tumorinhibitory factor (A. M. Ghaleb et al., Cell Res., 15, pp 92-96, 2005),and has known base sequences (NCBI accession Number NM_(—)010637(human), NM_(—)004235 (Mouse)). Klf1 gene, Klf2 gene, and Klf5 gene alsohave known base sequences (NCBI accession Number NM_(—)006563 (human),NM_(—)010635 (Mouse)), (NCBI accession Number NM_(—)016270 (human),NM_(—)008452 (Mouse)), and (NCBI accession Number NM_(—)001730 (human),NM_(—)009769 (Mouse)), respectively.

SOX9 gene is known as a transcription factor that regulates theexpression of, for example, type II collagen (V. Lefebvre et al., Mol.Cell. Biol. 17, pp 2336-2346, 1997), and has known base sequences (NCBIaccession Number NM_(—)000346 (human), NM_(—)011448 (Mouse)). ReplacingSOX9 gene with other SOX family gene leads to induction failure in thechondrocyte-like cells. Specifically, in the present invention,induction to the chondrocyte-like cells is only possible by thecombination of Myc family gene and/or Klf family gene with SOX9 gene,and it is important that these genes hold a combined, nondivisiblerelationship with each other.

These three genes commonly exist in mammals including humans, and any ofthese genes originating mammals may be used. Desirably, these genes areappropriately selected according to the origin of the recipient somaticcells. For example, the three genes are desirably of human origin whenthe somatic cells used originate in humans. Further, the three genes maybe wild-type genes, or mutated genes coding for mutated gene productsthat have the replacement, deletion, and/or insertion of several (forexample, 1 to 10, preferably 1 to 6, more preferably 1 to 4, furtherpreferably 1 to 3, particularly preferably 1 or 2) amino acids in theamino acid sequences, and that function in the same way as the wild-typegene products.

In the present invention, the three genes can be prepared according toan ordinary method, based on known sequence information. For example,the cDNA of the gene of interest can be prepared by extracting RNA frommammal-derived cells, followed by cloning using an ordinary method.

In the present invention, the genes introduced into the somatic cellsmay be a combination of SOX9 gene with at least one of the Myc familygene and the Klf4 family gene introduced as reprogramming factors.However, from the standpoint of improving the induction efficiency ofthe chondrocyte-like cells, the introduced genes are, for example,preferably a combination of SOX9 gene with at least one of Myc familygenes and at least one of Klf family genes; more preferably acombination of SOX9 gene with c-Myc gene or N-Myc gene, and Klf2 gene orKlf4 gene; particularly preferably a combination of three genes, i.e.,c-Myc gene, Klf4 gene, and SOX9 gene.

The two or more genes may be introduced into the somatic cells by usingmethods commonly used in animal cell transfections. Specific examples ofthe method that can be used to introduce the two or three genes into thesomatic cells include methods using vectors; calcium phosphate method;lipofection method; electroporation method; and microinjection method.For introduction efficiency, methods using vectors are preferable. Whenthe two or more genes are introduced into the somatic cells usingvectors, the vectors may be, for example, virus vectors, non-virusvectors, or artificial viruses. Considering safety, virus vectors suchas adenovirus and retrovirus are preferably used. Note that, whenvectors are used, the two or more genes may be incorporated intodifferent vectors, or may be incorporated in the same single vector.

Introducing the two or more genes into the somatic cells reprograms thesomatic cells, and induces the somatic cells into proliferativechondrocyte-like cells that have the properties of the chondrocytes. Thecells induced to chondrocyte-like cells can be selected from the somaticcells in which the two or more genes are introduced, based on thepresence or absence of cell proliferative ability and the presence orabsence of the properties comparable to the chondrocyte properties.Specifically, the chondrocyte-like cells can be selected from cellshaving a proliferative ability, using indices such as cell shape, thepresence or absence of specific staining for the chondrocytes, and thepresence or absence of chondrocyte marker gene expression in the cells.When the somatic cells include a reporter gene construct introducedtherein after being constructed by binding a drug resistant gene to thepromoter of a chondrocyte marker gene, cells that have acquired thecartilage properties can be selected by using cell growth in thepresence of a drug as an index, because such cells can grow in thepresence of a drug. Further, by taking advantage of the chondrocyte-likecells that assume a round or polygonal shape in a monolayer culture inliquid medium, these shapes also can be used as an index. Further,because the chondrocyte-like cells include glucosaminoglycanspecifically expressed in chondrocytes, the presence or absence ofglucosaminoglycan stained with alucian blue also can be used as anindex. Further, because the chondrocyte-like cells express chondrocytemarker genes (such as Col2a1, Acan, and SOX5), the presence or absenceof marker gene expression also can be used as an index.

The chondrocyte-like cells obtained as above can proliferate in amonolayer culture in liquid medium, stably grow generally up to about 9to 21 passages while maintaining the chondrocyte properties. Thechondrocyte-like cells can be cultured with media commonly used forculturing animal cells. DMEM medium containing about 1 to 25 volume %FBS is a preferred example of the medium used to culture thechondrocyte-like cells.

The thus-obtained chondrocyte-like cells, when applied to cartilagetissue in vivo, can form a new cartilage tissue of a three-dimensionalstructure using the cartilage tissue as a scaffold. When cultured invitro in the presence of a scaffolding material, the chondrocyte-likecells can form a cartilage tissue of a three-dimensional structure.

As described thus far, the chondrocyte-like cells obtained in thepresent invention have a proliferative ability, and can regeneratecartilage tissue in an organism. The chondrocyte-like cells are thuseffective for the treatment of cartilage disease such as osteochondrosisdeformans, chondrodystrophy arthritis (for example, rheumatoidarthritis), trauma, and osteonecrosis, and can be used as a cellpreparation (pharmaceutical composition) for cartilage tissueregeneration. The chondrocyte-like cells may be applied to a cartilagedisease site either alone or with a scaffolding material. When appliedto a cartilage disease site with a scaffolding material, thechondrocyte-like cells may be applied to the cartilage disease siteseparately from the scaffolding material. However, it is desirable thatthe chondrocyte-like cells and the scaffolding material be applied tothe cartilage disease site at the same time in the form of a cellpreparation, as will be described later.

When the chondrocyte-like cells are prepared as a cell preparation forcartilage tissue regeneration, a pharmaceutically acceptable carrier fordilution may be contained with the chondrocyte-like cells, as required.Examples of pharmaceutically acceptable carriers for dilution includephysiological salines, and buffers. Further, the cell preparation mayalso contain pharmacologically active components, and nutrient sourcecomponents for the chondrocyte-like cells, as required.

Desirably, the cell preparation contains a scaffolding material for thechondrocyte-like cells. When the cell preparation contains a scaffoldingmaterial, it is desirable that the chondrocyte-like cells be containedby being supported on the scaffolding material. The use of scaffoldingmaterial improves the graft rate of the chondrocyte-like cells at thediseased site of cartilage tissue, and further promotes cartilage tissueregeneration.

The scaffolding material is not particularly limited, as long as it ispharmaceutically acceptable. The scaffolding material is appropriatelyselected according to the target site of cartilage tissue. For example,gelatinous or porous, biodegradable or bioresorbable materials can beused. Preferred examples of scaffolding material include collagen,hydroxyapatite, α-TCP (tricalcium phosphate), β-TCP (tricalciumphosphate), polylactic acid, polyglycolic acid, and complexes of these.The scaffolding materials may be used either alone or in combinations oftwo or more. Of these scaffolding materials, collagen is preferable fromthe standpoint of efficient cartilage tissue regeneration. When collagenis used as scaffolding material, the collagen is desirably prepared intoa gel form of a three-dimensional structure.

The shape of the scaffolding material is not particularly limited, andis appropriately designed according to the shape of the damaged site ofthe cartilage tissue targeted by the cell preparation.

The chondrocyte-like cells can be supported on the scaffolding materialby, for example, inoculating or mixing the chondrocyte-like cells withthe scaffolding material, followed by culturing.

When the chondrocyte-like cells in the cell preparation are used bybeing supported on the scaffolding material or used to construct acartilage tissue of a three-dimensional structure, the proportion of thechondrocyte-like cells with respect to the scaffolding material may beappropriately set according to such factors as the site of the targetedcartilage tissue and the type of scaffolding material. As an example,the chondrocyte-like cells are used in a proportion of 1×10⁶ to 1×10⁸cells per 1 cm³ of the scaffolding material.

The method used to apply the cell preparation to the diseased site ofcartilage tissue is appropriately set according to such factors as thetype of cell preparation and the site of the targeted cartilage tissue.For example, the cell preparation may be directly injected through anincision at the diseased site of the treated cartilage tissue or thecell preparation may be injected to the diseased site of the treatedcartilage tissue using an arthroscope.

The dose of the cell preparation applied to the diseased site ofcartilage tissue may be appropriately set to an amount effective forcartilage tissue regeneration, based on such factors as the type of cellpreparation, the site of cartilage tissue, the extent of symptoms, andthe age and sex of a patient.

Further, the chondrocyte-like cells may be used to construct a cartilagetissue of a three-dimensional structure in vitro, and this construct maybe used as a cartilage tissue implant for the treatment of cartilagedisease that involves cartilage defects such as in osteochondrosisdeformans.

The chondrocyte-like cells can be used to construct a cartilage tissueof a three-dimensional structure by, for example, inoculating thechondrocyte-like cells in scaffolding material, and culturing the cellsin a medium capable of growing a chondrocyte-like cell until a cartilagetissue of a three-dimensional structure is constructed. Morespecifically, about 1×10⁶ to 1×10⁸ chondrocyte-like cells may beinoculated per 1 cm³ of scaffolding material, and cultured under 5% CO₂conditions at 37° C. for about 1 to 4 weeks. The same scaffoldingmaterial used for the cell preparation can be used to construct acartilage tissue of a three-dimensional structure. The shape of thescaffolding material may be appropriately set according to the shape ofthe implant of interest. The medium used to construct a cartilage tissueof a three-dimensional structure is not particularly limited, as long asit can grow the chondrocyte-like cells. For example, DMEM mediumcontaining about 1 to 25 volume % FBS may be used. From the standpointof clinical application, use of serum-free media of defined compositions(defined serum-free media) is desirable.

The thus-prepared cartilage tissue of a three-dimensional structureprepared as above is used as a cartilage tissue implant, either in thestate containing the scaffolding material, or after removing thescaffolding material.

The method used to apply the implant to the diseased site of thecartilage tissue is appropriately set according to such factors as theshape of the implant and the site of the targeted cartilage tissue. Forexample, the implant may be directly incorporated through an incision atthe diseased site of the treated cartilage tissue.

The chondrocyte-like cells also can form a cartilage tissue whenadministered to a site of an organism other than the cartilage tissue.Thus, the chondrocyte-like cells may be administered into the body of amammal, and the cartilage tissue formed by the chondrocyte-like cells inthe body of the mammal may be removed to obtain a cartilage tissueimplant.

The mammals used for the production of such cartilage tissue implantsmay be humans, or non-human mammals such as mice, rats, hamsters,rabbits, cats, dogs, sheep, pigs, cows, goats, and monkeys. Further, inthe production of the cartilage tissue implant, the administration siteof the chondrocyte-like cells is not particularly limited. However,considering the ease of the removal of the newly formed cartilagetissue, the administration site is preferably under the skin,particularly under the skin of the back. Further, in the production ofthe cartilage tissue implant, the chondrocyte-like cells may beadministered together with a scaffolding material, or alone without ascaffold. The chondrocyte-like cells can form a cartilage tissue of asufficient size in an organism without the administration of a scaffold.

In the production of the cartilage tissue implant, the dose of thechondrocyte-like cells for mammals is not particularly limited, and maybe generally about 10⁴ to 10⁸ cells, preferably about 10⁵ to 10⁷ cells.Formation of a cartilage tissue is recognized after 14 to 35 days,preferably after 21 to 28 days from the administration of thechondrocyte-like cells to mammals.

The cartilage tissue implant may be produced in the body of a cartilagedisease patient, and the cartilage tissue so produced may betransplanted into the cartilage disease site of the patient.Specifically, the chondrocyte-like cells may be administered to a siteof a cartilage disease patient other than the cartilage tissue, and anew cartilage tissue formed by the chondrocyte-like cells in the body ofthe patient may be removed and administered to the cartilage diseasesite of the patient for the graft treatment of cartilage disease.

Further, a non-human mammal including a cartilage tissue formed by thechondrocyte-like cells administered to the organism may be used as atool for evaluating the efficacy of a tested substance for the cartilagetissue. Specifically, a non-human mammal that includes a cartilagetissue formed by the chondrocyte-like cells may be administered with atested substance to determine and evaluate the efficacy of the testedsubstance for the cartilage tissue. As used herein, the “testedsubstance” refers to a substance to be evaluated for its efficacy forthe cartilage tissue. Specific examples include a candidate substance ofa therapeutic drug for cartilage disease.

Further, the chondrocyte-like cells can be used as a tool forelucidating the pathology of various cartilage diseases. Thechondrocyte-like cells induced from human somatic cells are useful as atool for the discovery and development of drugs for cartilage diseases.

2. Chondrocyte-Like Cell Preparation Composition

As described above, the chondrocyte-like cells can be prepared byintroducing into somatic cells at least one gene selected from the groupconsisting of Myc family gene and Klf family gene, in combination withSOX9 gene. The present invention thus provides a chondrocyte-like cellpreparation composition that includes SOX9 gene and at least one geneselected from the group consisting of Myc family gene and Klf familygene. The chondrocyte-like cell preparation composition includes a setof factors, namely, a reprogramming factor used to induce somatic cellsto chondrocyte-like cells and a cartilage-inducible transcriptionfactor. Desirably, the two or more genes are contained in a formintroducible into the somatic cells. A specific example is a vector thathas incorporated the two or more genes. The two or more genes may beincorporated in different vectors, or in the same single vector.

The types of the genes and vectors used for the chondrocyte-like cellpreparation composition are as described above.

EXAMPLES

The present invention is described below based on Examples. Note,however, that the invention is not limited by the followingdescriptions.

Example 1 Production of Chondrocyte-Like Cells from Dermal Fibroblastsand Embryonic Fibroblasts

1. Production of Col11a2-β Geo Transgenic Mice

Methods

First, transgenic mice were produced that express β-geo (fused gene ofβ-galactosidase gene and neomycin resistant gene) under the control ofCol11a2 promoter/enhancer sequences shown in FIG. 1, a, using thefollowing procedure.

742LacZInt, an α2 (XI) collagen gene-based expression vector, includes amouse Col11a2 promoter (−742 to +380), SV40 RNA splicing sites, aβ-galactosidase reporter gene, an SV40 polyadenylation signal, and a2.34-kb first intron sequence of Col11a2 as a enhancer (ReferenceLiterature 1). In order to produce a β-geo introducing gene, a 0.8-kbneomycin resistant gene fragment was ligated to the 3′-end of a 3.1-kbcDNA fragment that codes for LacZ. The β-geo fragment was incorporatedin a 742LacZInt expression vector at the Not I site by replacing theLacZ gene, and a Col11a2-β geo plasmid was produced.

The Col11a2-β geo plasmid was digested with EcoRI and PstI to releasethe inserts in the plasmid. The inserts were microinjected into thepronucleus of a F1 hybrid mouse (C57BL/6×DBA)-derived fertilized eggaccording to the method of Reference Literature 1 to produce atransgenic mouse. The transgenic mouse was identified by PCR assays ofthe genomic DNA extracted from the tail. Specifically, the transgenicmouse was identified by amplifying the genomic DNA by introducedgene-specific PCR, and by amplifying the 135-bp product specificallycontained in the β geo transgenic mouse, using a primer (CGC TAC CAT TACCAG TTG: SEQ ID NO: 1) that recognizes the LacZ gene, and a primer (CCAGTC ATA GCC GAA TAG: SEQ ID NO: 2) that recognizes the neomycinresistant gene. The transgenic mouse so identified was crossed withC57BL/6 mice for at least four generations.

The transgenic mouse prepared as above was studied by the X-gel stainingof the whole body and slices, according to the method described inReference Literature 2.

Results

The α2 (XI) collagen chain is a cartilage-specific matrix protein thatsupports the cartilage tissue structure, and has an important role inthe impact absorbing functions of the cartilage. It is known that theexpression of the Col11a2 promoter/enhancer sequences is cartilagespecific (Reference Literature 1). The Col11a2 promoter has an insulatoractivity, and is believed to contribute to stable expression of theintroduced gene in transgenic mouse. X-gal staining of the transgenicmouse showed LacZ activity specific to the chondrocytes, whereas noactivity was observed in other tissues (see the left diagram in FIG. 1,b). Further, histological analysis confirmed β geo expression in allchondrocytes (see the right diagram in FIG. 1, b).

2. Separation and Analyses of Mouse Embryonic Fibroblasts, Adult MouseDermal Fibroblasts, and Primary Chondrocytes from Col11a2-β GeoTransgenic Mice

Methods

Mouse embryonic fibroblasts (MEFs), adult mouse dermal fibroblasts(MDFs), and primary chondrocytes were isolated according to thefollowing procedure, using the transgenic mice obtained as above.

MEFs were separated according to the method of Reference Literature 3.Specifically, first, the head and the gut tissue were removed from a13.5 dpc embryo. The remaining body part was finely sliced, andtransferred to a tube after trypsin treatment. Cells were collected bycentrifugation, and suspended in DMEM medium that contained 10% FBS. Thecells (1×10⁶) were cultured in a 100-mm dish to obtain MEFs (firstpassage).

MDFs were prepared from the transgenic mouse, 3 to 6 months of age.Specifically, after shaving the transgenic mouse, the skin wassectioned, and subjected to trypsin treatment at 37° C. for 4 hours. Thefree cells were filtered through a nylon mesh (pore size, 40 μm; TokyoScreen, Tokyo, Japan) to produce a single-cell suspension, which wasthen cultured in a 100-mm dish to obtain MDFs (first passage).

The primary chondrocytes were separated according to the method ofReference Literature 4. Specifically, the transgenic mouse wasdissected, and the epiphyseal cartilages of the humerus and femur werecollected by separating the tissue in a DMEM medium that contained 2%FBS and streptomycin/penicillin. The cohesive tissue and the cartilagemembrane of the epiphyseal cartilage were physically removed afterdigestion with collagenase (type II, Sigma) at 37° C. for 30 min (2mg/ml in DMEM/2% FBS). After the removal of the cohesive tissue and thecartilage membrane, the epiphyseal cartilage was treated in acollagenase solution for 2 to 4 hours to free the primary chondrocytes.The free cells were collected by centrifugation (200×g at 4° C. for 5min), and suspended in a fresh medium. The cells were inoculated in a60-mm or 100-mm dish, and cultured in a 2% FBS-containing DMEM medium toobtain the primary chondrocytes.

Note that the first passages of MEFs and MDFs were cryopreserved inliquid nitrogen after the trypsin treatment, and later used for testing(described later).

The primary chondrocytes were evaluated for LacZ activity by X-galstaining.

The primary chondrocytes, MEFs, and MDFs were added to media thatcontained 0 to 900 μg/ma G418 (Geneticin), and incubated under 5% CO₂conditions at 37° C. to evaluate cell growth. Note that 2%FBS-containing DMEM medium was used for the primary chondrocyte culture,and 10% FBS-containing DMEM medium for the MEF and MDF cultures. Forcomparison, primary chondrocytes prepared from a wild-type littermatemouse using the same technique were also incubated in the presence ofG418 to evaluate cell growth.

Results

About 50% of the cells in the primary chondrocytes prepared from the βgeo transgenic mouse were stained by X-gal staining (see FIG. 1, c).This result suggests that the chondrocytes had dedifferentiated, orcontamination of the fibroblasts in the fibrous tissue attached to thecartilage had occurred during the preparation.

FIG. 1 in d shows the results of the incubation of the primarychondrocytes, MEFs, and MDFs in the presence of G418 after preparationfrom the β geo transgenic mouse. In contrast to the MEFs and MDFs thatdied out completely in the presence of 300 μg/ml G418, the primarychondrocytes prepared from the transgenic mouse grew even in thepresence of 900 μg/ml G418. The majority of the primary chondrocytesprepared from the wild-type F1 hybrid mouse (C57BL/6×DBA) died in thepresence of 300 μg/ml G418.

3. Assessment of Factors that Induce MEFs to Chondrocytes

Methods

The factors that induce somatic cells to chondrocytes were identified byevaluating the presence or absence of induction to chondrocytes throughtransformation of the MEFs using four reprogramming factors (Oct3/4,Sox2, c-Myc, and Klf-4), and a cartilage-inducible transcription factor(Sox9). Because cells showing a chondrocyte phenotype have resistance toG418, the test confirmed the presence or absence of induction tochondrocytes using G418 resistance as an index. Specifically, the testwas performed according to the following procedure.

In this test, the reprogramming factors and the cartilage-inducibletranscription factor were introduced into somatic cells using the retrovirus pMXs/Plat-E vector system as in the method of Reference Literature3. Specifically, the following retro virus vectors were used: A retrovirus vector having incorporated mouse c-Myc (pMXs-c-Myc), a retro virusvector having incorporated mouse Klf4 (pMXs-Klf4), a retro virus vectorhaving incorporated mouse Sox2 (pMXs-Sox2), and a retro virus vectorhaving incorporated mouse Oct3/4 (pMXs-Oct3/4). Human SOX9 wasincorporated in a retro virus vector by incorporating human SOX9 cDNA ina Gateway pENTR-1A vector (Invitrogen), and by inserting the resultingplasmid into pMXs-gw using LR reaction (Invitrogen).

The transcription factor was introduced to somatic cells according tothe following procedure. First, 8×10⁶ Plat-E cells were inoculated in a10-ml 10% FBS-containing DMEM medium (1 μg/ml puromycin, 10 μg/mlbrastcidine, supplemented with penicillin and streptomycin) in a 100-mmdish, and the Plat-E cells were transfected with each pMXs-based retrovirus vector on the next day, using Fugene 6 transfection reagent(Roche). The medium was exchanged 24 hours after the transfection.Twenty-four hours after the medium exchange, the medium was collected asa virus-containing supernatant from the Plat-E culture.

The cryopreserved MEFs were inoculated in a 100-mm dish. The MEFs orMDFs were subjected to trypsin treatment a day before transfection, and5×10⁵ cells in a 100-mm dish were statically cultured in 10%FBS-containing DMEM medium for 24 hours (third passage).

Each virus-containing supernatant obtained as above was then filteredthrough a 0.45-μm cellulose acetate filter (Schleicher & Schuell), andpolybrene (Nacalai Tesque) was added to the resulting filtrate at afinal concentration of 4 mg/ml to prepare a virus solution. Each virussolution was mixed according to the combination of transfecting genes toprepare a mixed virus solution. Note that each virus solution to bemixed in the preparation of the mixed virus solution was set so as tocontain the retro virus vectors in equal amounts.

The virus solution or virus mixture was then added to the cultured MEFdish, and incubated at 37° C. for 16 hours for transfection with theretro virus vector. After being incubated, the cells in the dish weretreated with trypsin, and statically cultured for 2 days in three 10-cmdishes that contained fresh DMEM medium supplemented with 10% FBS. Themedium was exchanged with 10% FBS-containing DMEM medium supplementedwith 500 μg/ml G418, and the cells were statically cultured for twoweeks while exchanging the medium with a medium of the same compositionevery other day.

The thus-cultured cells were stained with alucian blue, and then withcrystal violet, and the stained colonies in each dish were counted. Thenumber of stained colonies was measured by counting the total number ofstained colonies in the three dishes. Note that only the cellsdifferentiated into the chondrocytes are stained, because alucian bluestains the glucosaminoglycan specifically expressed in the chondrocytes,while crystal violet stains all the cells.

For comparison, MEFs were transformed using a retro virus vector(pMXs-EGFP) having incorporated GFP cDNA, and the transformed cells wereevaluated, using the same techniques described above.

Results

FIG. 2 shows the analysis results for the cells produced by introducingeach factor to MEFs. FIG. 2 in a represents colony numbers counted afterthe alucian blue staining and crystal violet staining of the cellsproduced by introducing each factor to MEFs. FIG. 2 b represents theresults of alucian blue staining and crystal violet staining for thecells obtained by transfecting the MEFs with the four reprogrammingfactors (Oct3/4, Sox2, c-Myc, and Klf-4) in combination with human SOX9.MEF transfection with human SOX9 alone did not induce colony formationin the presence of G418 (FIG. 2, a). In the transfection of MEFs onlywith the four reprogramming factors (Oct3/4, Sox2, c-Myc, and Klf-4),formation of small numbers of colonies of spindle-shaped cells ofnon-chondrocyte-like morphology was observed. However, these colonieswere not stained with alucian blue, and were not differentiatedchondrocytes. On the other hand, about 110 G418-resistant colonies per10-cm dish were observed in the transfection of MEFs with the fourreprogramming factors in combination with SOX9, and about 30% of thesecolonies were stained with alucian blue (see FIGS. 2, a and b). Thecells obtained by transfecting the MEFs with the four reprogrammingfactors in combination with SOX9 had shapes that varied from colony tocolony, some being colonies of polygonal cells (left in FIG. 2,c)—similar in shape to the primary chondrocytes (FIG. 1, c)—, and otherbeing colonies of spindle shaped cells (right in FIG. 2, c), as with theMEFs (FIG. 2, d).

In order to identify the importance of each factor on the formation ofG418-resistant colonies, MEFs were transfected with three of the fourreprogramming factors and Sox9, and the resulting cells were analyzed.FIG. 2 in e represents colony numbers counted after the alucian bluestaining and crystal violet staining of the cells to which threereprogramming factors and Sox9 were introduced. The average colonynumber was found to decrease in the absence of c-Myc or Klf4. On theother hand, the colonies did not decrease even without the introductionof Oct3/4 or Sox2. These results suggest that the transfection by c-Myc,Klf4, and Sox9 is important for the induction of MEFs to chondrocytes.In the transduction of MEFs with c-Myc, Klf4, and Sox9, about 50% ofabout 250 G418-resistant colonies were formed by polygonal cells ofchondrocyte-like morphology (see FIG. 2, f).

4. Assessment of Factors that Induce MDFs to Chondrocytes

Methods

MDFs were transfected with various combinations of the fourreprogramming factors (Oct3/4, Sox2, c-Myc, and Klf-4) and thecartilage-inducible transcription factor (SOX9) using the foregoingtechnique, and the properties of the resulting cells were analyzed.

Results

FIG. 3 represents the analysis results for the cells obtained byintroducing each factor to MDFs. FIG. 3A to C represents colony numberscounted after the alucian blue staining and crystal violet staining ofthe cells produced by introducing each factor to MDFs, and the number ofcolonies of polygonal cells. MDF transfection with SOX9 alone did notinduce colony formation in the presence of G418 (see FIG. 3, A). Onlysmall numbers of colonies were formed in the transfection of MDFs onlywith the four reprogramming factors (Oct3/4, Sox2, c-Myc, and Klf-4). Onthe other hand, about 120 G418-resistant colonies resulted from the MDFstransduced with the four reprogramming factors (Oct3/4, Sox2, c-Myc, andKlf-4) in combination with SOX9, and about 30% of these colonies wereformed by polygonal cells of chondrocyte-like morphology. These resultsobtained from MDFs had the same tendency as the results obtained fromMEFs (see FIG. 2, a).

The colony number slightly decreased when any of the c-Myc, Klf4, andOct3/4 were lacking from the four reprogramming factors (Oct3/4, Sox2,c-Myc, and Klf-4) in the presence of SOX9, whereas the colony numberincreased when Sox2 was lacking (see FIG. 3, B). Colonies formed byround or polygonal cells were not observed in the colonies when eitherc-Myc or Klf4 was lacking from the four reprogramming factors in thepresence of SOX9 (FIG. 3, B), and about ⅕ of the colonies were formed byround or polygonal cells when Oct3/4 was lacking from the fourreprogramming factors in the presence of SOX9 (see FIG. 3, B). Theseresults suggest that c-Myc, Klf4, and SOX9 are important in theformation of colonies that include G418-resistant cells of achondrocyte-like shape from MDFs. Specifically, it was found that thecombination of c-Myc, Klf4, and SOX9 produced about 200 G418-resistantcolonies from MDFs, and that about 40% of these colonies were formed byround or polygonal cells (see FIG. 3, C). The combination of c-Myc andSOX9 also formed about 200 G418-resistant colonies. Although most ofthese colonies were formed by spindle-shaped or more flat cells ofnon-chondrocyte-like morphology, colonies formed by round or polygonalcells of chondrocyte-like morphology were also observed, though in smallnumbers (see FIG. 3, C). Further, about 350 G418-resistant colonies wereformed with the combination of only SOX9 and Klf4, and these coloniesincluded colonies formed by small numbers of round or polygonal cells ofchondrocyte-like morphology (see FIG. 3, C).

As demonstrated by the foregoing results, the introduction of c-Myc,Klf-4, and SOX9 created about 50 colonies of G418-resistant cells ofchondrocyte-like morphology from 5×10⁵ MDF cells. Adding Oct3/4 to thecombination of these three factors did not influence the number ofcolonies formed by the G418-resistant cells of chondrocyte-likemorphology. Colony formation was impeded by addition of Sox2. Further,formation of G418-resistant colonies by the cells of chondrocyte-likemorphology was also observed with both the combination of c-Myc andSOX9, and the combination of Klf-4 and SOX9. The results also suggestedthat the Sox2 introduction inhibits the induction to the cells ofchondrocyte-like morphology.

On the other hand, formation of G418-resistant colonies was not observedwhen SOX5 and SOX6, used in place of SOX9, were introduced to MDFs withc-Myc and Klf-4 using the same technique. SOX5 and SOX6 are known tohave supportive action for SOX9, but do not have the transactivationdomain present in SOX9. Considering this, the transactivation domainpresent in SOX9 is likely to be involved in the induction to the cellsof chondrocyte-like morphology.

Genes belonging to the Myc family and the Klf family basically have thesame biological activity distinct to the family. The foregoingexperimental results thus show that the use of the genes of the Mycfamily and the genes of the Klf family in combination with SOX9 enablesinduction of somatic cells to the cells of chondrocyte-like morphology.

5. Clone Production Methods

The following eleven colonies were selected from the G418-resistantcolonies induced from the MDFs, and clones were produced.

-   -   One from the colonies produced by the transfection with c-Myc,        Klf-4, Sox2, Oct3/4 and Sox9 (clones will be denoted as MKSO-1)    -   Two from the colonies produced by the transfection with c-Myc,        Klf4, Sox2, and Sox9 (clones will be denoted as MKS-1 or MKS-2)    -   Four from the colonies produced by the transfection with c-Myc,        Klf4, Oct3/4, and Sox9 (clones will be denoted as MKO-1 to        MKO-4)    -   Four from the colonies produced by the transfection with c-Myc,        Klf4, and Sox9 (clones will be denoted as MK-1 to MK-4)

Each target colony was subjected to trypsin treatment, and cells werecollected. The cells are cultured in a 10% FBS-containing DMEM mediumsupplemented with 500 μg/ml G418 under 5% CO₂ conditions at 37° C. for 6to 10 days in a 96-well plate. The cells proliferated in the 96-wellplates were transferred to a 24-well plate, and cultured under 5% CO₂conditions at 37° C. for 24 to 31 days. The proliferated cells in the24-well plate were transferred to a 6-well plate, and cultured under 5%CO₂ conditions at 37° C. for 18 to 31 days. The cells were transferredto a 10-cm dish, and the cells at this stage were defined as a fourthpassage. The proliferated cells were cultured in a DMEM mediumsupplemented with 500 μg/ml G418 and 10% FBS, and subcultured every sixdays.

Results

Upon culturing the cells contained in the eleven colonies usingG-4,8-containing medium according to the foregoing method, the MKO-4colony-derived cells stopped proliferating after the seventh passage.After culturing, ten clones (MKSO-1, MKS-1, MKS-2, MKO-1 to MKO-3, andMK-1 to MK-4) but MKO-4 were produced. Each clone was polygonal inshape, and had the morphology of chondrocytes (see FIG. 3, E).

6. Evaluation of Cloned Cell Properties

The cloned cells were analyzed by alucian blue staining, introduced-geneexpression analysis, chondrocyte marker gene expression analysis,karyotyping, gene expression pattern analysis, the analysis ofmethylated CpG dinucleotide in the Col1a2 promoter region, and theanalysis of proliferative properties.

6-1. Analysis by Alucian Blue Staining

The cloned cells (sixth passage), and MDFs (third passage) were culturedin a 10% FBS-containing DMEM medium supplemented with 500 μg/ml G418 ina 60-mm dish, and further cultured for 14 days after confluence. Thecultured cells were then subjected to alucian blue staining.

FIG. 4A shows the results. The cultured cells were strongly stained withalucian blue, verifying the presence of glycosaminoglycan. Note that thestrength of staining varied among the clones.

6-2. Introduced-Gene Expression Analysis

Expression of the introduced genes in the cloned cells was analyzed byRT-PCR and western blotting, using primers that amplify the transcriptsderived from the genes introduced to retro virus, but do not amplify thetranscripts of the endogenous genes. Specifically, the analysis wasperformed according to the following procedure.

The cloned cells (sixth passage) and MDFs (third passage) were culturedin a 10% FBS-containing DMEM supplemented with 500 μg/ml G418, and theprimary chondrocytes (first passage) prepared from β geo transgenicmouse were cultured in a 2% FBS-containing DMEM medium, each in a 60-mmdish. After confluence, total RNA in the cells was extracted usingRNeasy Mini Kits (Qiagen, Santa Clarita, Calif.). The extracted totalRNA was digested with DNase, and contaminating genomic DNA was removed.The resulting total RNA (1 μg) was then reverse-transcribed into asingle-stranded (first-strand) cDNA using QuantiTect ReverseTranscription (Qiagen). The resulting cDNA (2 μl) was PCR amplified in amixture (20 μl) containing ExTaq (Takara Bio) and primers (4 pmol)specific to each gene, and the RNA expression level of each gene wasmeasured. Table 1 below lists the primers used.

TABLE 1 Target gene Primers used Sequence Klf4 Klf4 Tg RT SGACCACCTTGCCTTACACA Klf4 Tg RT AS CCCTTTTTCTGGAGACTAAAT c-Mycc-Myc Tg RT S TCGCTACCATTACCAGTTG c-Myc Tg RT AS CCCTTTTTCTGGAGACTAAATOct3/4 Oct3/4 Tg RT S TCCCATGCATTCAAACTG Oct3/4 Tg RT ASCCCCTGTTGTGCTTTTAATC Sox2 Sox2 Tg RT S CCATTAACGGCACACTGC Sox2 Tg RT ASCCTTACGCGAAATACGGG hS0X9 SOX9 RT S CCAGCGAACGCACATCAA SOX9 RT ASGGAGTTCTGGTGGTCGGTGTA

The cloned cells (sixth passage) and MDFs (third passage) were culturedin a 10% FBS-containing DMEM supplemented with 500 μg/ml G418, and theprimary chondrocytes (first passage) prepared from β geo transgenicmouse were cultured in a 2% FBS-containing DMEM medium, each in a 60-mmdish. The cells were lysed after confluence. The cell lysate wassubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), followed by electroblotting and immunostaining. Theantibodies used are anti-Sox9 antibodies (Santa-Cruz Biotechnology,Inc., 1:200 dilution), anti-c-Myc antibodies (Santa-Cruz Biotechnology,Inc., 1:200 dilution), anti-Klf4 antibodies (Santa-Cruz Biotechnology,Inc., 1:200 dilution), anti-Oct3/4 antibodies (Santa-Cruz Biotechnology,Inc., 1:600 dilution), anti-Sox2 antibodies (Santa-Cruz Biotechnology,Inc., 1:200 dilution), and anti-β-actin antibodies (Cell SignalingTechnology, 1:5,000 dilution).

FIG. 4B shows the results of RT-PCR analysis, and FIG. 4C shows theresults of western blotting. Expression of the introduced genes in theclones was confirmed from the results of RT-PCR analysis. Westernblotting confirmed expression of the introduced genes in the clonescells at the protein level, but did not confirm expression of thesegenes in MDFs.

6-3. Chondrocyte Marker Gene Expression Analysis

Expression of chondrocyte marker genes in the cloned cells was analyzedby RT-PCR. Specifically, total RNAs were obtained from the cloned cells(sixth passage), MDFs (third passage), and the primary chondrocytes(first passage) prepared from β geo transgenic mouse using the foregoingtechnique, and the expression of chondrocyte marker genes (Col2a1, Acan,Hapln1, Sox5, Sox6, Col9a1, Col9a2, Col9a3, Col11a1, Col11a2) and MDFmarker genes (Col1a1, Col1a2, Gapdh, RT-) was analyzed by RT-PCRanalysis. The primers used are listed in Table 2.

TABLE 2 Target gene Primers used Sequence Gapdh Gapdh RT SGAGATGATGACCCTTTTGGCT Gapdh RT AS TCAAGGCCGAGAATGGGAAG Sox5 Sox5 RT SCCCCTCAAAGCCTCTGTC Sox5 RT AS CTTGCTGCTCTCGCCTGA Sox6 Sox6 RT STCATCCCGGCCTAAGACA Sox6 RT AS ACAGGGCAGGAGAGTTGAG Col2a1 Col2a1 RT STTGAGACAGCACGACGTGGAG Col2a1 RT AS AGCCAGGTTGCCATCGCCATA Col11a1Col11a1 RT S ATGAGTATGCACCTGAGGAT Col11a1 RT AS GGAGTCTCAGTCTGGTAAGGTTCol11a2 Col11a2 RT S GACTGTAAGAAGCGAGTTACC Col11a2 RT ASGCCTTCAAAGACTTCATCG Col9a1 Col9a1 RT S TGTAGACTTCAGGATTCCAACCol9a1 RT AS CCAAATGTTCCAGTGCTT Col9a2 Col9a2 RT S TGGAAGGGAGTGCGGATTCol9a2 RT AS CGACCAGGATCACCCAGAAT Col9a3 Col9a3 RT S TGGTGTGCCGGGACTTGATCol9a3 RT AS CACCCAGCTCGCCAGTTCTA Col1a1 Col1a1 RT SGCAACAGTCGCTTCACCTAC Col1a1 RT AS GTGGGAGGGAACCAGATTG Col1a2 Col1a2 RT STCGGGCCTGCTGGTGTTCGTG Col1a2 RT AS TGGGCGCGGCTGTATGAGTTCTTC AcanAcan RT S CCCTCGGGCAGAAGAAAGAT Acan RT AS CGCTTCTGTAGCCTGTGCTTG

FIG. 5 a represents the results of the chondrocyte marker geneexpression analysis, and FIG. 5 b represents the results of the MDFmarker gene expression analysis. The results showed that the clonedcells were capable of expressing the chondrocyte marker genes at variouslevels. MKS-1, MKO-2, MK-1, MK-3, and MK-4 expressed the chondrocytemarker genes, whereas MKS-2 and MK-2 did not express these genes. It wasalso confirmed that MKS-1 expressed fibroblast-specific type I collagengenes (Col1a1 and Col1a2).

Further, the presence of LacZ-negative cells in the primary chondrocytesprepared from β geo transgenic mouse raises the possibility that theperifibrous tissue-derived fibroblasts adhered to the cartilage werecontaminated (see FIG. 1, c). This might explain the detection of thetype I collagen gene (Col1a1 and Col1a2) mRNA, thought to be expressedin fibroblasts but not in pure chondrocytes, from the primarychondrocyte-derived RNA.

6-4. Karyotyping

The cloned cells were karyotyped by quinacrine-Hoechst staining. Theanalysis was performed at the International Council for LaboratoryAnimal Science (ICLAS) Monitoring Center (Japan).

FIG. 5 c represents a part of the results. MKS-2, MKO-2, and MK-4 hadnormal 40XY karyotypes, whereas MK-3 was a mixture of normal 40XY and41XY+4.

6-5. Gene Expression Pattern Analysis

The overall gene expression pattern of the cloned cells

(MKS-1, MKO-2, MKI-1, MK-3, MK-4), MDFs, and the primary chondrocytesprepared from β geo transgenic mouse was analyzed as follows, usingscatter plots in DNA microarray analysis.

A biotin-labeled cRNA was obtained from 250 ng of total RNA, using aMessage Amp III RNA Amplification Kit (Ambion). Ten micrograms of thefragmented cRNA were then hybridized against an Affymetrix 430 2.0 GeneChip array at 45° C. for 16 hours. The DNA chip was washed, and stainedfurther. The resulting DNA chip was scanned using Affymetrix Fluidicsstation 450 and a scanner, and the resulting image was analyzed usingGCOS software. For normalization, calculations were performed using MAS5.0 algorithm. Cluster analyses were performed using Cluster 3.0 (TheUniversity of Tokyo).

The results of scatter plots in the DNA microarray analysis arepresented in FIGS. 6, a to c. The number of overexpressed genes wassmaller in the MDFs as measured against the primary chondrocytes,whereas the number of overexpressed genes was greater in the primarychondrocytes as measured against the MDFs (see FIG. 6, a). This resultcoincides with the implication that the primary chondrocytes werecontaminated with the fibroblasts. Further, the number of overexpressedgenes in MK-3 measured against the primary chondrocytes (see FIG. 6, b)was smaller than that in MK-3 measured against the MDFs (see FIG. 6, c).Further, the number of overexpressed genes in the primary chondrocytesmeasured against MK-3 (see FIG. 6, b) was about the same as that in MDFsmeasured against MK-3 (see FIG. 6, c). This is considered to be due tothe contamination of the primary chondrocytes with the fibroblasts.These results suggest that MK-3 is similar to pure chondrocytes at theoverall transcription level. In both MK-3 and primary chondrocytes, theexpression levels of the cartilage matrix genes including Col2a1,aggrecan gene (Acan), and Col9a1 were considerably higher than theexpression levels of the other genes (see FIG. 6, b).

The results of the cluster analyses are presented in FIG. 6, d. Itbecame clear from the results of cluster analyses that the cloned cellsother than MKS-1 were classified in different clusters from those of theMDFs, primary chondrocytes, and MKS-1. This coincides with the RT-PCRfinding that MKS-1 expresses both chondrocyte marker genes and MDFmarker genes, and with the implication that the primary chondrocytes arecontaminated with the fibroblasts.

6-6. Analysis of Methylated CpG Dinucleotide in Promoter Regions ofChondrocyte Marker Genes and MDF Marker Genes

The methylation status of the cytosineguanine (CpG) dinucleotide in thepromoters of the chondrocyte marker genes (Col2a1 and Acan) and in thepromoter of the MDF marker gene (Col1a2) were evaluated for the clonedcells (MK-3, MK-4) and MDFs using bisulfite genomic sequencing analyses.Specifically, bisulfite genomic sequencing analyses were performed usingthe following technique. A bisulfite treatment was performed using anEpiTect Bisulfite kit (Qiagen) according to the protocol attached to thekit. Table 3 below lists the PCR primers used. The amplificationproducts were cloned into pMD20-T vector using a Mighty TA-cloning Kit(Takara). Ten clones randomly selected for each gene were then sequencedusing T7 and T3 primers.

TABLE 3 Target promoter Primers used Sequences Col1a2 Col1a2-Me-S2GGATTGGATAGTTTTTGTTTTT promoter Col1a2-Me-AS2  AAAACCCAAACCTACCTTATTTAcan Acan-Me-S2 GGTGTTAGAGGGGTTTATAGAGTTGAG promoter GA Acan-Me-AS2CTCCTCCAAAAACTTCAATCCTTTATC CCTAC Col2a1 Col2a1-Me-S3TAGAGGGGGTAGTGTGGTAGTT promoter Col2a1-Me-AS3 CCCTCATACAAAAAACCCTAAAA

The results are presented in FIG. 6, e. The cytosineguanine (CpG)dinucleotide in the Col1a2 promoter was highly methylated in MK-3 andMK-4, but was not methylated in MDFs. With regard to the methylationstatus of the CpG dinucleotide in the Col2a1 and Acan promoters, therewas essentially no methylation in both the cloned cells (MK-3, MK-4) andMDFs.

6-7. Analysis of Proliferative Properties

The cloned cells (sixth passage) and MDFs (sixth passage) were culturedin a 10% FBS-containing DMEM medium in a 60-nm dish, and theproliferative properties were evaluated.

The results are presented in FIG. 7, a. MKO-2, MK-1, MK-3, and MK-4showed exponential growth for at least 48 days, and spindle-shaped orflat cells started to appear after 40 days from the start of culturing.On the other hand, the MDFs stopped growing after 15 days from the startof culturing. MKS-1 showed a rapid increase in growth rate, andunderwent a morphological change to a spindle shape after 24 days fromthe start of culturing. This suggests that MKS-1 dedifferentiation hadoccurred, and may be a reflection of abnormal chromosome number in thecells.

Some of the cells of each clone were separated after the cell numberexceeded 1×10¹⁰ cells, and cultured by being inoculated in a 10-cm dish.The cells were further cultured for days after confluence. The cellswere then stained with alucian blue. The results of alucian bluestaining are presented in FIG. 7, b. It became clear from the resultsthat the strength of alucian blue staining was stronger in thechondrocyte-like cells than in MDFs, and that the chondrocyte-like cellsretained the chondrocyte characteristics even after the cells hadreached a certain number.

7. Production of Cartilage Tissue

The cloned cells (MK-3) and MDFs were used to produce cartilage tissueusing the following technique.

Collagen gel culture was performed using a collagen gel culture kit(Nitta Gelatin Inc.) according to the protocol attached to the kit.First, the chondrocyte-like cells (MK-3) and MDFs were digested withtrypsin/EDTA. The cells were then added to a 0.25% type I acid dissolvedcollagen solution prepared at 4° C., and suspended in 2×10⁷ cells/ml.The cell suspension (500-μl liquid droplet) was then added to the centerof each well of a 6-well plate, and was gelled at 37° C. The resultinggel-cell complex was covered with DMEM medium that contained 10% FBS (3ml), and cultured under 5% CO₂ conditions at 37° C. The medium exchangedwith a fresh medium every other day. After being cultured for 3 weeks,the gel-cell complex was fixed with 10% formaldehyde, and embedded inparaffin. A part of the gel-cell complex treated as above was thenstained with alucian blue and nuclear fast red. Further, a part of thegel-cell complex was treated with primary antibodies against type IIcollagen (goat-derived polyclonal antibodies; Santa-Cruz Biotechnology,Inc.; 1:200 dilution), washed, and further treated with secondaryantibodies (Alexa Fluor 488 Rabbit Anti-goat IgG; Invitrogen).

The results are presented in FIG. 7, c. Histological analysis of theMK-3 three-dimensional culture product in type I collagen gel confirmeda tissue structure of a small cavity configuration surrounded bysubstances stained with alucian blue, revealing the formation of acartilage-like tissue in the gel-cell complex. The MK-3-containinggel-cell complex showed immunoactivity against the anti-type II collagenantibodies, whereas such immunoactivity was not observed in theMDF-containing gel-cell complex.

8. Cartilage Tissue Production In Vivo

Cells of chondrocyte-like morphology (MK-5; chondrocyte-like cells)induced by introducing c-Myc, Klf4, and SOX9 to MDFs were cloned usingthe foregoing technique. Note that GFP was also introduced to MK-5 usinga retro virus vector having incorporated GFP cDNA.

The chondrocyte-like cells (MK-5) were digested with trypsin/EDTA. Thecells were then added to a DMEM medium containing 10 volume % FBS, andsuspended in 1×10⁷ cells/ml to prepare a cell suspension. The cellsuspension (0.1 mL) was subcutaneously injected to the back of a nudemouse (6 weeks of age, female, BALB/cA Jc1-nu/nu).

The fluorescence at the back of the mouse was observed after 4 weeksfrom the administration of the cell suspension. GFP-expressing clumpswere observed under the skin injected with the MK-5 cell suspension(FIGS. 8, A and B). The cell suspension-injected site was removed, fixedwith 4% formaldehyde, and embedded in paraffin. The continuous tissueslice so treated was subjected to safranine O staining, andimmunostained with anti-GFP antibodies. The results are presented inFIG. 8, C. The subcutaneous adipose tissue of the MK-5-injected mousecontained tissues of cells scattering in the matrix stained red withsafranine O, showing the formation of a cartilage tissue under the skinof the nude mouse. The GFP positive cells are believed to represent theinjected chondrocyte-like cells, and the range of these cells completelycoincided with the region of the cartilage tissue stained with safranineO. This suggests that all of the survived injected MK-5 cellsdifferentiated into chondrocytes and formed a cartilage tissue. FIG. 8Drepresents magnifications of the rectangles shown in FIG. 8, C. Theresult demonstrated that the chondrocyte-like cells were capable offorming a cartilage tissue in the absence of scaffolds, and could be putto practical applications for the regeneration of cartilage tissue.

9. Summary Discussion

It became clear from the foregoing results that the introduction ofc-Myc, Klf-4, and Sox9 in combination can produce cells(chondrocyte-like cells) that have a proliferative ability and theproperties of the chondrocytes. It was confirmed that thechondrocyte-like cells so produced were actually capable of forming acartilage tissue of a three-dimensional structure when cultured with acollagen gel, or by being directly administered to an organism.

Example 2 Formation of Cartilage Tissue from Chondrocyte-Like CellsMethods

Eleven chondrocyte-like cells (MK-5 to MK-15) were obtained bytransfecting the MDFs with the c-Myc, Klf4, and Sox9 genes using themethod of Example 1. Two of these chondrocyte-like cell lines (MK-7 andMK-10) were digested with trypsin/EDTA, and suspended in a DMEM mediumcontaining 10 volume % FBS to prepare a cell suspension (1×10⁷cells/ml). 0.1 ml of the cell suspension was subcutaneously injected tothe back of a nude mouse (female, 6 weeks of age, BALE/cA Jc1-nu/nu).The injection site was removed in week 16 post-injection in the MK-7cell-injected mouse, and in week 8 post-injection in the MK-10cell-injected mouse. The removed sites were fixed with 4%paraformaldehyde, and embedded in paraffin. Then, tissue slices wereproduced, and stained with safranine O, fast green, and ironhaematoxylin.

Results

The results are presented in FIG. 9. The subcutaneous adipose tissue ofthe MK-7 cell- and MK-10 cell-injected mice contained tissues of cellsscattering in the matrix stained red with safranine O, confirming theformation of a cartilage tissue under the skin of the nude mouse. Notumor formation was recognized at the MK-7 cell- or MK-10 cell-injectedsite.

These results confirmed that the method of the present invention can beused to obtain chondrocyte-like cells without forming tumors for atleast 16 weeks.

Example 3 Analysis of Genomic DNA of Chondrocyte-Like Cells Results

Experiments were conducted to evaluate the identity of thechondrocyte-like cells (MK-1, MK-3, and MK-4) obtained in Example 1, andof the chondrocyte-like cells (MK-5, MK-7, MK-10, and MK-15) obtained inExample 2, as follows.

First, genomic DNA was obtained from the chondrocyte-like cells using anordinary method, and fragmented by digestion with EcoRI and BamHI. Thegenomic DNA fragments were developed by electrophoresis on agarose gel,transferred to a nylon membrane, and subjected to southern hybridizationusing Klf4 cDNA probes.

The results are presented in FIG. 10. As can be seen in FIG. 10, thechondrocyte-like cells obtained in Examples 1 and 2 showed differentband patterns for different cell lines, each independently representingan established cell line.

Example 4 Production of Chondrocyte-Like Cells from AdiposeTissue-Derived Stromal Cells Methods

Adipose tissue-derived stromal cells (ADSCs) were separated from thesubcutaneous adipose tissue using the method of Reference Literature 5.Specifically, a piece of subcutaneous adipose was removed from aCol11a2-β geo transgenic mouse, 3 to 6 months of age, prepared in themanner described in Example 1. The tissue was sectioned, and treatedwith 0.2% collagenase at 37° C. for 2 to 4 hours. The liberated cellsafter the collagenase treatment were filtered through a nylon mesh (poresize, 70 μm; Tokyo Screen, Tokyo, Japan). The separated cells werecollected by centrifugation (200×g, 4° C., 10 min). The cells weresuspended in a fresh 5% FBS-containing DMEM medium, and centrifugedagain (200×g, 4° C., 10 min) to collect cells. The collected cells werecultured in a 60-mm or 100-mm dish to obtain ADSCs (first passage).

Thereafter, c-Myc, Klf-4, and Sox9 were introduced into the subcutaneousadipose cells, and cultured in a 5% FBS-containing DMEM mediumcontaining 500 μg/ml G418 (10 ml), using the method of Example 1.

The cells treated as above were stained with alucian blue and crystalviolet, and cell shapes were observed, as in Example 1.

For comparison, a retro virus vector prepared by incorporating GFP cDNAin pMXs vector was used to transform the subcutaneous adipose, and thetransformed cells were evaluated, using the technique described above.

Results

The results are presented in FIG. 11. Formation of about 380G418-resistant colonies was observed per 10-cm dish after theintroduction of c-Myc, Klf-4, and Sox9 to ADSCs. About 60% of thesecolonies were stained with alucian blue. Further, about 20% of thestained colonies were round or polygonal cells of chondrocyte-likemorphology. No colony formation was recognized in the GFP-introducedadipose-derived stromal cells.

These results strongly suggested that the introduction of c-Myc, Klf-4,and Sox9 to the subcutaneous adipose tissue-derived cells could formchondrocyte-like cells that have a proliferative ability and theproperties of the chondrocytes, as with the case of MEFs and MDFs inExample 1.

Example 5 Production of Chondrocyte-Like Cells from Human-Derived DermalFibroblasts Methods 1. Plasmid Preparation

A lentivirus vector system was used to introduce genes to human-deriveddermal fibroblasts. LR Clonase II plus reaction (Invitrogen) was used toprepare a human c-MYC-incorporated lentivirus vector (pLe6-CMVp-hc-MYC),a human KLF4-incorporated lentivirus vector (pLe6-CMVp-hKLF4), a humanOCT3/4-incorporated lentivirus vector (pLe6-CMVp-hOCT3/4), and a humanSOX9-incorporated lentivirus vector (pLe6-CMVp-F(−)hSOX9).

2. Cell Preparation

Neonatal normal human dermal fibroblasts (NHDF) were purchased fromLonza (product code: CC-2511). For use, NHDFs were maintained in 10%FBS-containing DMEM medium.

3. Virus Infection

293FT cells (6×10⁶ cells; Invitrogen) were transfected with 3 μg of eachlentivirus vector and 9 μg of Virapower packaging mix (Invitrogen),using Lipofectamine 2000 (Invitrogen). After 48 hours, the transfectantsupernatant was collected, and filtered through a 0.45-μm celluloseacetate filter (Whatman). Polybrene (Nacalai Tesque) was then added tothe obtained filtrate in a final concentration of 4 mg/ml to prepare avirus solution. A mixed virus solution was prepared by mixing the virussolutions so as to contain equal amounts of Le6-CMVp-hc-MYC,pLe6-CMVp-hKLF4, pLe6-CMVp-hOCT3/4, and pLe6-CMVp-F(−)hSOX9.

A day before the transfection, NHDFs (5×10⁵ cells) were inoculated in10% FBS-containing DMEM medium in a 100-mm dish. The medium was removedfrom the 100-mm dish, and the cells were incubated at 37° C. for 16hours with addition of the mixed virus solution for transfection withthe lentivirus vectors. After being incubated, the cells in the dishwere treated with trypsin, and statically cultured in four 10-cm dishesthat contained fresh 10% FBS-containing DMEM medium. After thetransfection, the cells were statically cultured for 10 days whileexchanging the medium with a medium of the same composition every otherday. The cultured cells were then stained with alucian blue.

For comparison, an EGFP cDNA-incorporated lentivirus vector was used totransform NHDFs, and the transformed cells were evaluated, using thetechniques described above.

Results

The results are presented in FIG. 12. Cell death was observed in many ofthe NHDFs to which the three reprogramming factors (OCT3/4, C-MYC, andKLF-4) and SOX9 were introduced. This suggests that the introduction ofthese reprogramming factors to NHDFs induces cell death in many cells.

The survived cells after the introduction of the three reprogrammingfactors (OCT3/4, C-MYC, and KLF-4) and SOX9 to NHDFs formed colonies.Some of these colonies were strongly stained with alucian blue. In theculture dish of the EGFP-introduced NHDFs, the cells did not die, andproliferated. The EGFP-introduced NHDFs were not stained with alucianblue (see FIG. 12, a). The cells contained in the colonies obtainedafter culturing with the introduced three reprogramming factors (OCT3/4,C-MYC, and KLF-4) and SOX9 had a morphology (see FIG. 12, b) that moreresembled the morphology of human primary chondrocytes (see FIG. 12, d)than that of the EGFP-introduced cells (see FIG. 12, c).

These results strongly suggested that the technique of the presentinvention can be used to also induce human NHDFs to chondrocyte-likecells that have a proliferative ability and can form a cartilage tissue.

A LIST OF REFERENCE LITERATURES

-   Reference Literature 1: N. Tsumaki, T. Kimura, Y. Matsui et al., J.    Cell Biol. 134 (6), 1573 (1996).-   Reference Literature 2: A Nagy, M Gertsenstein, K Vintersten et al.,    Manipulating the Mouse Embryo., 3rd ed. (Cold Spring Harbor    Laboratory Press, New York, 2003).-   Reference Literature 3: K. Takahashi and S. Yamanaka, Cell 126 (4),    663 (2006).-   Reference Literature 4: A. Aszodi, E. B. Hunziker, C. Brakebusch et    al., Genes Dev. 17 (19), 2465 (2003).-   Reference Literature 5: Bjorntorp, P. et al. Isolation and    characterization of cells from rat adipose tissue developing into    adipocytes. J. Lipid Res. 19, 316-324 (1978).

SEQUENCE LISTING

PCT_chondrocyte-like cells, and _(—)20091218_(—)104843_(—)3.txt

1. A chondrocyte-like cell producing process comprising the step ofintroducing into a somatic cell a SOX9 gene and at least one geneselected from the group consisting of Myc family gene and Klf familygene.
 2. A producing process according to claim 1, wherein the Mycfamily gene is a c-Myc gene.
 3. A producing process according to claim1, wherein the Klf family gene is a Klf4 gene.
 4. A producing processaccording to claim 1, wherein the somatic cell originates in humans. 5.A producing process according to claim 1, wherein the somatic cell is adermal fibroblast or an adipose tissue-derived stromal cell.
 6. Achondrocyte-like cell obtained by introducing into a somatic cell a SOX9gene and at least one gene selected from the group consisting of Mycfamily gene and Klf family gene.
 7. A cell preparation for cartilagetissue regeneration, comprising the chondrocyte-like cell of claim
 6. 8.A cell preparation according to claim 7, wherein the cell preparationincludes a scaffolding material.
 9. A cell preparation according toclaim 8, wherein the scaffolding material is a collagen.
 10. An implantcomprising a cartilage tissue constructed by using the chondrocyte-likecell of claim
 6. 11. A process for producing a cartilage tissue implant,the process comprising the steps of: administering the chondrocyte-likecell of claim 6 into a body of a mammal; and removing a cartilage tissueformed from the chondrocyte-like cell in the body of the mammal.
 12. Acartilage disease therapeutic method, the method comprising the stepsof: administering the chondrocyte-like cell of claim 6 into a cartilagedisease patient at a non-cartilage tissue site; and removing a cartilagetissue formed from the chondrocyte-like cell, and transplanting theremoved cartilage tissue to the cartilage disease site of the patient.13. A use of the chondrocyte-like cell of claim 6 for the production ofa cell preparation for cartilage tissue regeneration.
 14. A useaccording to claim 13, wherein the cell preparation for cartilage tissueregeneration is a therapeutic agent for cartilage disease.
 15. A use ofa composition containing the chondrocyte-like cell of claim 6 and ascaffolding material for the production of a cell preparation forcartilage tissue regeneration.
 16. A use according to claim 15, whereinthe scaffolding material is a collagen.
 17. A non-human mammal forming acartilage tissue, wherein the non-human mammal is produced byadministering the chondrocyte-like cell of claim 6 into a non-humanmammal, and by forming a cartilage tissue from the chondrocyte-like cellin a body of the mammal.
 18. A method for determining the efficacy of atested substance for a cartilage tissue, the method comprising the stepof administering the tested substance to the non-human mammal of claim17 and determining the efficacy of the tested substance for thecartilage tissue.
 19. A chondrocyte-like cell preparation composition,comprising a SOX9 gene and at least one gene selected from the groupconsisting of Myc family gene and Klf family gene.